|M R||Endogenous||55||Rabbit IgG|
Western blot analysis of extracts from various cell lines using TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific).Learn more about how we get our images.
Western blot analysis of extracts from L-929 cells, transfected with 100 nm SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® TNF-R1 siRNA I (Mouse Specific) #13369 (+), or SignalSilence® TNF-R1 siRNA II (Mouse Specific) #13570 (+), using TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific) confirms the silencing of the TNF-R1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse TNF-R1 (mTNF-R1; +), using TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific) recognizes endogenous levels of total mouse TNF-R1 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of mouse TNF-R1 protein.
TNF-α is an important cytokine produced by numerous cell types including neutrophils, activated lymphoctyes, macrophages and NK cells. It plays a critical role in inflammatory responses and in apoptosis (1). TNF-α exists as a membrane-anchored and soluble form, both of which show biological activity. Response to TNF-α is mediated through two receptors, TNF-R1, which is widely expressed, and TNF-R2, which is expressed mainly in immune and endothelial cells (2). Antagonists to TNF-α have been validated as therapeutic targets for rheumatoid arthritis and other immune disorders (3).
The two receptors for TNF-α, TNF-R1 (55 kDa) and TNF-R2 (75 kDa) can mediate distinct cellular responses (4,5). In most cases cytotoxicity elicited by TNF has been reported to act through TNF-R1 (6,7). Cytotoxicity is mediated by a "death domain" with the intracellular region of the receptor that binds to the death domain adaptor protein TRADD and triggers the activation of caspases (8). Soluble forms of both receptors have also been characterized which can bind TNF-α and may play an important role in immune disorders (9,10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalSilence is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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